1. Field of the Invention
This invention relates generally to the field of blood coagulation and specifically to new stable recombinant meizothrombin-like molecules which have anti-coagulant activity.
2. Description of Related Art
During the final stages of blood coagulation, prothrombin is converted from an inactive zymogen to the serine protease thrombin, an enzyme which plays a central role in hemostasis. Prothrombin is synthesized in the liver and undergoes several post-translational modifications prior to secretion. These modifications include glycosylation, cleavage of the pre- and pro-peptides and vitamin K-dependent .gamma.-carboxylation of the first 10 amino terminal glutamic acid residues. The .gamma.-carboxyglutamic acid (Gla) residues are involved in the metal ion-dependent interaction of prothrombin with phospholipid surfaces. Activation of prothrombin to thrombin results from the proteolytic cleavage of Arg.sup.271 --Thr.sup.272 and Arg.sup.320 --Ile.sup.321 by factor Xa. Although factor Xa alone will catalyze the activation of prothrombin slowly, the reaction progresses five orders of magnitude more rapidly in the presence of the prothrombinase complex, which consists of factor Xa, the cofactor factor Va, calcium ions and a negatively charged phospholipid surface.
Depending on the order of peptide bond cleavage, two intermediate products, meizothrombin and prethrombin-2, can exist in the reaction pathway (See FIG. 1). Meizothrombin is produced by proteolytic cleavage of the Arg.sup.320 --Ile.sup.321 bond by factor Xa yielding fragment 1.2-thrombin A chain joined to the thrombin B chain by a disulfide bond. Meizothrombin is capable of catalyzing the cleavage of the Arg.sup.155 --Ser.sup.156 bond which releases the fragment 1 (F1) domain and yields an active species called meizothrombin (desF1), which no longer contains the .gamma.-carboxylated (Gla) region and exhibits functional activities different from those of meizothrombin. The pair, fragment 1.2 plus prethrombin-2, results from the cleavage of the Arg.sup.271 --Thr.sup.272 bond. Although prethrombin-2, with the exception of the bond at Arg.sup.320 --Ile.sup.321, is identical in covalent structure to thrombin, it has no proteolytic activity.
The existence of meizothrombin as an intermediate in the prothrombinase-catalyzed activation of prothrombin was described by Rosing, et al., (J. Biol. Chem. 261:4224, 1986). Further studies indicated that it appears to be the main, if not sole intermediate of the activation of prothrombin by the fully assembled prothrombinase complex in vitro. Human thrombin formed on the surface of endothelial cells influences the formation of meizothrombin via a feedback mechanism, leading to the accumulation of meizothrombin (desF1) in the final phase of prothrombin activation. The specific role of factor Va in meizothrombin formation has not been clarified to date. Factor Va, however, interacts with both factor Xa and prothrombin and presents them to one another in the formation of a ternary enzyme-substrate-cofactor complex (Boskovic, et al., J. Biol. Chem. 265:10497, 1990). In addition, if factor Va is omitted from the reaction, prethrombin-2 is the main intermediate observed.
Studies of human meizothrombin have been hampered by its extremely transient existence due to autolysis that results in rapid formation of meizothrombin (desF1), and further rapid activation to .alpha.-thrombin. Furthermore, electrophoretic analysis under non-reducing conditions is complicated because meizothrombin and meizothrombin (desF1) have molecular weights that are identical to those of prothrombin and prethrombin-1. Reversible thrombin inhibitors allow the isolation of meizothrombin, but make subsequent enzymatic characterization difficult. Even in the presence of the reversible inhibitor dansylarginine N-(3-ethyl-1,5 pentanediyl) amide (DAPA), the meizothrombin generated is only stable for a few hours on ice before autolysis occurs and meizothrombin (desF1) appears.
The study of the role of native meizothrombin in thrombosis and hemostasis disorders has been severely hampered by its unstable nature. For example, two congenital dysprothrombinemia cases have been described, prothrombin Barcelona and prothrombin Madrid. In both cases, the defect is characterized by a very low thrombin generation and coagulant activity, but normal prothrombin antigen level. The molecular defect in both families is due to the substitution of Arg.sup.271 to Cys which disrupts one of the two factor Xa cleavage sites and alters the activation of the molecule. Exposure of prothrombin Barcelona and Madrid to factor Xa results in cleavage of the Arg.sup.320 --Ile.sup.321 bond, yielding meizothrombin. This mutant prothrombin appears to offer a model to study the activity of meizothrombin, but since the thrombin cleavage sites are intact, generation of meizothrombin (desF1) and .alpha.-thrombin (desA 1-13) would probably occur and complicate the analysis. In addition, the new cysteine residue could also form an abnormal disulfide bond and affect the folding of the protein.
Presently, one of the most common anticoagulant used in patients is heparin. Heparin acts as an anticoagulant by a heparin-antithrombin interaction, which induces a conformational change in antithrombin III (ATIII) that is responsible for acceleration of inactivation of clotting enzymes. Heparin is a heterogeneous mixture of sulfated polysaccharide chains ranging in molecular weight from 5000-35000 daltons. Many studies have shown that the low molecular weight fraction (LMW) of heparin is less hemorrhagic than unfractionated heparin (UF) (Esquivel, et al., Thromb. Res. 28:389, 1982; Carter, et al., Blood 59:1239, 1982). However, other studies have found that for equivalent antithrombin doses, some LMW fractions have the same hemorrhagic effect as UF heparin (Diness, et al., Thromb. Haemost. 55:410, 1986).
Thus, there exists a need for a anticoagulant agent which inhibits coagulation with greater specificity and without the degree of hemorrhagic side effects of heparin. It would be especially valuable if such anticoagulant agent could be used in clinical settings where the thrombosis process is unresponsive to heparin treatment. The apparently unique functional properties of native meizothrombin and the difficulties associated with this transient prothrombin activation intermediate motivated the investigation for a more stable form of the protein. The present invention fulfills a longfelt need for a safe, stable meizothrombin-like anticoagulant molecule.